Nanoparticle-mediated gene delivery, genomic editing and ligand-targeted modification in various cell populations

ABSTRACT

An improved nanoparticle for transfecting cells is provided. The nanoparticle includes a core polyplex and a silica coating on the core polyplex and, optionally, a polymer attached to an outer surface of the silica coating, where the polyplex includes an anionic polymer, a cationic polymer, a cationic polypeptide, and a polynucleotide. Also provided is an improved method of modifying intracellular polynucleotides. The method includes contacting a cell with a nanoparticle that includes a core polyplex and a silica coating on the core polyplex and, optionally, a polymer attached to an outer surface of the silica coating, where the polyplex includes an anionic polymer, a cationic polymer, a cationic polypeptide, and a polynucleotide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application and claims prioritybenefit of commonly owned and co-pending U.S. patent application Ser.No. 15/024,264, filed Mar. 23, 2016, which is a National stageapplication of PCT/US2014/057000, filed on Sep. 23, 2014, which claimspriority under 35 U.S.C. § 119 to U.S. Provisional Application No.61/881,072, filed Sep. 23, 2013, the entire disclosures of each of theprior applications are hereby incorporated herein by reference.

GOVERNMENT RIGHTS STATEMENT

This invention was made with U.S. Government support under R01 AG030637awarded by the National Institutes of Health. The U.S. Government hascertain rights in the invention.

BACKGROUND OF THE INVENTION Technical Field

The present invention generally relates to use of nanoparticles totransfect cells. More particularly, the present invention relates tocoated nanoparticles with a polyplex core for intracellular delivery ofploynucleotides to modify gene expression.

Background Information

Introducing polynucleotides into cells to alter gene expression requiresappropriate packaging of the polynucleotides to protect them fromdegradation before cell entry, to permit entry into cells, and to directdelivery to the appropriate subcellular compartment. Effectiveness inaltering expression may also depend on time-frames of release ofpolynucleotides from packaging after cellular entry. Availablenanoparticle-based technologies for modifying gene expression sufferfrom low levels of cellular transfection and limited effectiveness upontransfection, at least in part because of their limitations insatisfying the foregoing requirements. It is therefore desirable toobtain a nanoparticle-based transfection agent and method of use thereofthat addresses all of these requirements to enhance effectiveness.

SUMMARY OF THE INVENTION

The shortcomings of the prior art are overcome, and additionaladvantages are provided, through the provision, in one aspect, of ananoparticle. The nanoparticle includes a core polyplex and a silicacoating on the core polyplex, and the polyplex includes an anionicpolymer, a cationic polymer, a cationic polypeptide, and apolynucleotide. In another aspect, the nanoparticle may also include apolymer attached to an outer surface of the silica coating.

A method of modifying intracellular polynucleotides is also provided.The method includes contacting a cell with a nanoparticle that includesa core polyplex and a silica coating on the core polyplex, and thepolyplex includes an anionic polymer, a cationic polymer, a cationicpolypeptide, and a polynucleotide. In another aspect, the nanoparticlemay also include a polymer attached to an outer surface of the silicacoating.

Additional features and advantages are realized through the techniquesof the present invention. These, and other objects, features andadvantages of this invention will become apparent from the followingdetailed description of the various aspects of the invention taken inconjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

One or more aspects of the present invention are particularly pointedout and distinctly claimed as examples in the claims at the conclusionof the specification. The foregoing and other objects, features, andadvantages of the invention are apparent from the following detaileddescription taken in conjunction with the accompanying drawings inwhich:

FIGS. 1A-1B are diagrammatic representations of some embodiments of ananoparticle and components thereof in accordance with an aspect of thepresent invention;

FIG. 2A is a diagrammatic representation of how a nanoparticle may bemanufactured in accordance with an aspect of the present invention;

FIG. 2B is a diagrammatic representation of means by which a cell mayuptake and intracellularly process a nanoparticle in accordance with anaspect of the present invention;

FIG. 3 is a graph illustrating the effects on polyplex complexation ofincluding different ratios of various charged polymers andpolynucleotides in accordance with an aspect of the present invention;

FIG. 4 is a graph illustrating the effects on polyplex complexation ofincluding different ratios of various charged polymers andpolynucleotides, with or without including an anionic polymer in thepolyplex, in accordance with an aspect of the present invention;

FIG. 5 is a graph illustrating the destabilizing effect on a polyplex ofincluding increasing amounts of an anionic polymer in the presence orabsence of cationic polypeptides in accordance with an aspect of thepresent invention;

FIG. 6 is a graph illustrating sizes of nanoparticles possessing variouslayers in accordance with an aspect of the present invention;

FIG. 7 is photomicrographs of cells transfected with variousnanoparticles demonstrating cellular uptake and subcellular localizationof nanoparticles following transfection in accordance with an aspect ofthe present invention;

FIG. 8 is photomicrographs of cells transfected with nanoparticlesshowing duration of residence of nanoparticles in cells followingtransfection in accordance with an aspect of the present invention;

FIGS. 9A-B is photomicrographs showing cellular uptake of nanoparticlespossessing a layer of polymers attached to the outside of a silicacoating of a polyplex in accordance with an aspect of the presentinvention;

FIG. 10 is a diagrammatic representation of TALEN peptides encoded forby a nucleic acid included in a nanoparticle that cause knockdown ofexpression of sclerostin in accordance with an aspect of the presentinvention;

FIGS. 11A-11C are graphs illustrating the effects transfecting cellswith different amounts of nanoparticles that target sclerostinexpression on sclerostin and β-catenin expression in accordance with anaspect of the present invention;

FIGS. 12A-12F are graphs illustrating the effects of transfecting cellswith different amounts of nanoparticles that target sclerostinexpression on expression levels of various cellular signaling peptidesin accordance with an aspect of the present invention;

FIG. 13 is photomicrographs demonstrating effects of transfecting cellswith nanoparticles that target sclerostin expression on expression of aco-transfected reporter gene that is responsive to transcription factorswhose activity is inhibited by sclerostin-mediated signalling inaccordance with an aspect of the present invention;

FIGS. 14A-14C are photomicrographs demonstrating effects of transfectingcells with nanoparticles that target sclerostin expression onmineralization in accordance with an aspect of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Aspects of the present invention and certain features, advantages, anddetails thereof, are explained more fully below with reference to thenon-limiting embodiments illustrated in the accompanying drawings.Descriptions of well-known materials, fabrication tools, processingtechniques, etc., are omitted so as to not unnecessarily obscure theinvention in detail. It should be understood, however, that the detaileddescription and the specific examples, while indicating embodiments ofthe invention, are given by way of illustration only, and are not by wayof limitation. Various substitutions, modifications, additions and/orarrangements within the spirit and/or scope of the underlying inventiveconcepts will be apparent to those skilled in the art from thisdisclosure.

The present disclosure provides, in part, a multilayered nanoparticlefor transfecting cells with agents to modify gene expression.Nanoparticles designed for improved serum stability, targetted deliveryto specific cell types, greater nuclear specificity andcompartment-specific unpackaging, improved ability to retain significantpayload levels during initial stages of internalization, and ability tomaintain release of payload for a various durations followinginternalization, and methods of use thereof, are provided.

In one aspect, complexes of polynucleotides with polymers, orpolyplexes, created by condensation of cationic polymers andpolynucleotides in the presence of anionic polymers may mediateincreased transfection efficiency over polynucleotide-cationic polymerconjugates. Though this process may produce more particles and increasethe net surface area of nanoparticles exposed for cellular uptake, animproved electrostatic repulsory element may also be at play inreleasing nucleic acids through this technique. Surprisingly, incontrast to a more rapid disaggregation of nucleotides from nanoparticlepolyplexes that include anionic polymers as would have been predicted onthe basis of existing literature, in one aspect of the presentinvention, including an anionic polymer in a nanoparticle polyplex coremay prolong the duration of intracellular residence of the nanoparticleand release of agents that affect gene expression or otherwise regulatecellular function, or payloads.

In another aspect, the presence of a cationic polypeptide in ananoparticle may mediate stability, subcellular compartmentalization,and payload release. As one example, fragments of the N-terminus ofhistone peptides, referred to generally as histone tail peptides, withinvarious polyplexes are not only capable of being deprotonated by varioushistone modifications, such as in the case of histoneacetyltransferase-mediated acetylation, but may also mediate effectivenuclear-specific unpackaging as components of polyplexes. Theirtrafficking may be reliant on alternative endocytotic pathways utilizingretrograde transport through the Golgi and endoplasmic reticulum, andthe nature of histones existing inside of the nuclear envelope suggestsan innate nuclear localization sequence on histone tail peptides. In oneaspect of the present invention, including a histone tail peptide maypromote nuclear localization of nanoparticles and result inenzyme-mediated release of polynucleotide payload therefrom.

In another aspect, silica coatings of polyplexes may seal their payloadsbefore and during initial cellular uptake. Commonly used polyplexesconsisting of poly(ethylenimine) and DNA have a tendency to shed themajority (˜90%) of their payloads during cellular internalization, withthe remaining payload often remaining bound to its cationicnanocarrier's polymeric remains. With transiently stabilizinginterlayers of silica, greater intracellular delivery efficiency may beobserved despite decreased probability of cellular uptake. In anotheraspect of the present invention, coating a nanoparticle polyplex with asilica coating may seal the polyplex, stablizing it until its releaseupon processing in the intended subcellular compartment.

In another aspect of the present invention, transfection efficiency maybe further increased by adding another layer of cationic polymer, makingthe delivery efficiency as much as two orders of magnitude greater thana bare or silica-coated polyplex, presumably due to the anionic natureof an oligomeric silica coating being cell repulsive. In a furtheraspect, silica-coated polyplexes and their further-layered derivativesare stable in serum and are suitable for in vivo experiments unlikecationic polymer/nucleic acid conjugates on their own.

FIGS. 1A-1B show examples of components of a nanoparticle in accordancewith the present invention. In accordance with the present invention, ananoparticle polyplex core may include a polynucleotide, an anionicpolymer, a cationic polymer, and a cationic polypeptide. A silicacoating may then be applied to the polyplex core, and polymers may thenbe attached to an outer surface of the silica coating. Thepolynucleotide may be a DNA vector for driving intracellular expressionof a nucleic acid sequence it contains. However, a nanoparticle may alsocomprise other types of polynucleotides, such as linear DNA or varioustypes of RNA, including dsDNA, ssDNA, mRNA, siRNA, or CRISPR RNAsequences, or others, or any combination of the foregoing. Ananoparticle may also include, in addition to or in place of any of theforegoing examples of polynucleotides, a peptide nucleic acid, othercharged or polar small molecules between 50 and 1000 Da, oralternatively between 200 and 10 kDa, in size, such as cyclicnucleotides such as cAMP, DNA origami templates, aptamers, chargedpolypeptides, proteins or protein fragments between 2 and 100 kDa,peptoids, phosphorylated or sulfated constituents, anionically modifiedconstituents, and multimeric or oligomeric combinations of theforegoing. A person of ordinary skill would understand any of theforegoing, or any combination thereof, as being included within thepresent invention.

Continuing with FIG. 1A, in one aspect of the invention, a cationicpolymer within the polyplex may be a polypeptide containing cationicamino acids and may be, for example, poly(arginine), poly(lysine),poly(histidine), poly(ornithine), poly(citrulline), or a polypeptidethat comprises any combination of more than one of the foregoing. Ananoparticle may also include, in addition to or in place of any of theforegoing examples of cationic polymers, poly(ethylenimine),poly(aspartamide), polypeptoids, a charge-functionalized polyester, acationic polysaccharide, an acetylated amino sugar, chitosan, or avariant or variants that comprise any combination of more than one ofthe foregoing, in linear or branched forms.

In one example, a cationic polymer may comprise a poly(arginine), suchas poly(L-arginine). A cationic polymer within the polyplex may have amolecular weight of between 1 kDa and 200 kDa. A cationic polymer withinthe polyplex may also have a molecular weight of between 10 kDa and 100kDa. A cationic polymer within the polyplex may also have a molecularweight of between 15 kDa and 50 kDa. In one example, a cationic polymercomprises poly(L-arginine) with a molecular weight of approximately 29kDa, as represented by SEQ ID NO: 1 (PLR). In another example, acationic polymer may comprise linear poly(ethylenimine) with a molecularweight of 25 kDa (PEI). In another example, a cationic polymer maycomprise branched poly(ethylenimine) with molecular weight of 10 kDa. Inanother example, a cationic polymer may comprise branchedpoly(ethylenimine) with a molecular weight of 70 kDa. In anotherexample, a cationic polymer may comprise a D-isomer of poly(arginine) orof any of the foregoing polymers such as polypeptides, which may beparticularly advantageous because polymers such as polypeptidescontaining a D-isomer may be less susceptible to degradation within acell and therefore have a prolonged effect on influencing payloadrelease and the rate thereof over time.

Continuing with FIG. 1A, in a further aspect of the invention, ananionic polymer within the polyplex may be a polypeptide containinganionic amino acids, and may be, for example, poly-glutamic acid orpoly-aspartic acid, or a polypeptide that comprises any combination ofthe foregoing. A nanoparticle may also include, in addition to or inplace of any of the foregoing examples of anionic polymers, aglycosaminoglycan, a glycoprotein, a polysaccharide, poly(mannuronicacid), poly(guluronic acid), heparin, heparin sulfate, chondroitin,chondroitin sulfate, keratan, keratan sulfate, aggrecan,poly(glucosamine), or an anionic polymer that comprises any combinationof the foregoing. In one example, an anionic polymer may comprisepoly-glutamic acid. An anionic polymer within the polyplex may have amolecular weight of between 1 kDa and 200 kDa. An anionic polymer withinthe polyplex may also have a molecular weight of between 10 kDa and 100kDa. An anionic polymer within the polyplex may also have a molecularweight of between 15 kDa and 50 kDa. In one example, an anionic polymeris poly(glutamic acid) with a molecular weight of approximately 15 kDa.Polymers consisting of or including a D-isomer of glutamic acid may beparticularly advantageous because they may be less susceptible todegradation within a cell and therefore have a prolonged effect oninfluencing payload release and the rate thereof over time. For example,the anionic polymer within the polyplex may have the sequencerepresented by SEQ ID NO: 2 (PDGA). In another example, an anionicpolymer may comprise a D-isomer of any of the foregoing polymers orpolypeptides, which may be particularly advantageous because polymerssuch as polypeptides containing a D-isomer may be less susceptible todegradation within a cell and therefore have a prolonged effect oninfluencing payload release and the rate thereof over time. Continuingwith FIG. 1A, in another aspect of the invention, a cationic peptide ina nanoparticle's polyplex core may be a fragment of a histone peptide,such as of the H1, H2, H3, or H4 proteins. The fragment may includeamino acids whose sequence corresponds to the N-terminus of a histoneprotein. For example, the fragment may comprise up to the first 5 (SEQID NO: 9), 10 (SEQ ID NO: 10), 15 (SEQ ID NO: 11), 20 (SEQ ID NO 12), 25(SEQ ID NO: 13) or more N-terminal amino acids of a histone protein. Thefragment may also be amidated on its C-terminus. The fragment may alsohave been modified such that one or more lysine residue is methylated,one or more histidine, lysine, arginine, or other complementary residuesare acetylated or susceptible to acetylation as a histoneacetyltransferase or acetyl CoA substrate, or any combination of theforegoing. For example, a cationic peptide in a nanoparticle polyplexcore may have the sequence as represented by SEQ ID NO: 3, whichcomprises the first 25 amino acids of the human histone 3 protein,amidated on its C-terminus, and tri-methylated on the lysine 4 inaccordance with the present invention (HTP).

In another embodiment, a nanoparticle may include or contain, inaddition to or in place of any of the foregoing cationic polypeptides, anuclear localization sequence. A cationic polypeptide may comprise anuclear localization sequence on its N- or C-terminus. A nuclearlocalization sequence may comprise an importin or karyopherin substrate,or may have or contain a sequence corresponding to SEQ ID NO: 8. Inanother embodiment, a nanoparticle may include, in addition to or inplace of any of the foregoing cationic polypeptides, a mitochondriallocalization signal or a peptide fragment of mtHSP70.

Continuing with FIG. 1B, in another aspect of the invention, thenanoparticle may comprise a reversible coating that provides stabilityto the polyplex core prior to cellular or compartmental internalization,preventing premature degradation or destabilization. For example, asilica coating may be applied to the polyplex core. In another example,calcium phosphate or hydroxyapatite may be applied to a polyplex core.In another example, a branched cationic polymer, polypeptide, or peptoidmay be applied to a polyplex core, with an anionic charge excess. Acoating, such as a silica coating, may protect the polyplex fromdegradation before exposure to the endosomal microenvironment.

In another aspect, a nanoparticle may comprise a layer of polymersattached to or electrostatically bound with the external surface ofcoated polyplex, such as to or with the external surface of a silicacoating. Such external polymers may serve to prevent cellular repulsionof the coated polyplex so as to promote contact with and uptake by acell. An external polymer layer may also serve to promoteinternalization by specific cell types, such as if the externallyattached polymer is or mimics a ligand to a receptor expressed by a celltype of which transfection is desired. A polymer in a polymer layerattached to the outer surface of coating on a polyplex may be frombetween 0.1 to 20 kDa in size, or may be up to 40 or 50 kDa in size.

Examples of polymer comprising a polymer layer attached to the externalsurface of the coated core polyplex include those represented by SEQ IDNO: 4, which is an approximately 10 kDa poly(arginine) polymer, and SEQID NO: 5, which is human vasoactive endothelial growth factor protein,in accordance with the present invention. In another example, a polymercomprising a layer attached to the external surface of the coated corepolyplex may comprise an anchor substrate of from between 1 to 25repeating anionic or cationic moieties at the N-terminus, C-terminus,5′, or 3′ end of a polymer, polypeptide, or polynucleotide to provideelectrostatic conjugation of a targeting motif contained in the polymer,polypeptide, or polynucleotide to the coated polyplex core. In anotherexample, a polymer comprising a layer attached to the external surfaceof the coated core polyplex may comprise a polymer, polypeptide, orpolynucleotide sequence that exhibits base pair complementarity orbinding affinity for an amino acid sequence binding motif to bindadditional layers that may be added thereupon.

In another aspect of the present invention, illustrated in FIG. 2A, acationic polyplex is created, then coated with a silica coating.Polyplex cores of nanoparticles may be created via electrostaticinteractions leading to condensation. Two equal-volume solutions may becreated, one with pH-unadjusted 40 mM HEPES (pH˜5.5) combined with 0.1%w/v a cationic polymer and a cationic polypeptide in water and the otherwith 30 mM Tris-HCl (pH˜7.4) combined with 0.1% w/v anionic polymers anda polynucleotide in water. In one embodiment, the cationic polymercomprises SEQ ID NO: 1, the anionic polymer comprises SEQ ID NO: 2, andthe cationic polypeptide comprises SEQ ID NO: 3. These solutions may becombined via dropwise addition of the cationic solution to the anionicone with no stirring. After 30 minutes of incubation at roomtemperature, a 200 uL solution containing 10 ug of nucleic acids withinpolyplexes may be added dropwise to a 45 mM sodium silicate (Sigma)solution in Tris-HCl (pH=7.4) and allowed to incubate for between 8 and24 hours at room temperature. Silica-coated polyplexes may be isolatedvia centrifugation with a 300 kDa Nanosep® filter (Pall, PortWashington, N.Y.) at 3000 g in order to isolate complexes from unboundsilica species and polymers. Nanoparticles may further be resuspended ina solution containing a polymer to be attached to the external surfaceof the silica coating. For example, they may be resuspended in asolution comprising a polymer represented by SEQ ID NO: 4 or SEQ ID NO:5 at 0.1% w/v for one hour. Nanoparticles may then be centrifuged againbefore resuspension in transfection medium. This method is but oneexample of manufacturing nanoparticles in accordance with the presentinvention.

FIG. 2B is a diagrammatic representation of contacting a cell with ananoparticle in accordance with the present invention leading tocellular internalization of the nanoparticle, such as bycaveolae-mediated endocytosis or macropinocytosis. Nanoparticles mayfurther be retrogradely transported through the Golgi and endoplasmicreticulum or processed through lysosomal pathways, resulting in loss ofthe coating, such as a silica coating, and exposure of the polyplexcore. The polyplex core may further be translocated into the cellnucleus, where enzymatic processing my degrade the cationic polymer,such as through activity of arginases, or otherwise promote unpackagingof the polyplex core, such as through acetylation of a histone tailpeptide within the polyplex, leading to release of polynucleotides suchas plasmid DNA from the polyplex core, in accordance with the presentinvention. Other intracellular processing steps modifying theconstituents of a nanoparticle and its polyplex core or coating thereofor polymer layer attached to the coating may also occur in accordancewith the present invention.

In a further aspect, the present invention includes optimized ratios ofanionic and cationic polymers, cationic polypeptides, andpolynucleotides for complexation of a polyplex core as part of ananoparticle. In one example, plasmid DNA was fluorescently tagged withethidium bromide (40 ng EtBr/ug DNA) before addition of variouspolymeric constituents in molar [1(positive)]:[1(negative)] ratios of[amine (n)]:[phosphate (p)+carboxylate (c)], or of c:p in the instanceof poly(D-glutamic acid) (PDGA; SEQ ID NO: 2) addition. Addition oflinear poly(ethylenimine) (PEI, 25 kDa) was compared to addition ofpoly(L-arginine) (PLR, 29 kDa; SEQ ID NO: 1) independently, as well asin conjunction with a H3K4(Me3) histone tail peptide (HTP; SEQ ID NO:3), in order to quantify similar complexation behaviors between the twopolymers as part of a binary complex (i.e., PEI+DNA or PEI+DNA) orternary complexes (HTP+PEI+DNA or HTP+PLR+DNA). Where a cationic polymerand cationic polypeptide were both present, the relative molar ratio ofeach component was 60%:40%, respectively. A Zeiss filter andspectrophotometer were used to excite EtBr-tagged DNA at 510 nm for anemission at 595 nm, and results were compared amongst variousformulations with unbound EtBr as a negative control.

FIG. 3 is a graph showing the effects of varying the ratio of anionic orcationic polymers or polypeptides to polynucleotides. The X axis showscharged polymer-to-phosphate ratio and the Y axis shows relativefluorescence following combination of indicated constituents. A decreasein relative fluorescence indicates displacement of EtBr from DNA andpolyplex formation. Ratios of cationic polymer, or of cationic polymerand cationic polypeptide, to DNA of approximately 5:1 and higherexhibited an approximately 40% decrease in fluorescence indicatingcomplexation of DNA and polymers into polyplexes. Addition of PDGA inthe absence of cationic polymers or cationic polypeptides did not affectcomplexation.

After complexing PLR-HTP-DNA, PEI-HTP-DNA, PLR-DNA and PEI-DNApolyplexes and determining that PDGA possesses no ability to causecomplexation of polynucleotides, PDGA's influence on formation kineticswas established by comparison of [5.5(positive)]:[1(negative)] and[10(positive)]:[1(negative)] molar ratios of [amine (n)]:[phosphate (p)]and [amine (n)]:[phosphate (p)+carboxylate (c)] on complexationefficiencies in order to determine effects of excess cationic andequalized charge ratios on nanoparticle complexation. Inclusion ofcarboxylate groups from PDGA was expected to have effects on overallformation kinetics comparable to inclusion of phosphate groups from DNA.Relative fluorescence was compared to DNA without addition of polymersor polypeptides or EtBr in the absence of DNA as controls.

FIG. 4 indicates the effects of adding PDGA to cationic polymers andcationic polypeptides on polyplex complexation kinetics. DNA wascomplexed with HTP, PLR or PEI, with or without addition of PDGA. Shownare experiments using cationic polymer (PLR or PEI)-to-polynucleotidemolar ratios of 5.5:1 (as shown in the bars labeled n/p=5.5) andcationic polymer (PLR or PEI)-to-polynucleotide plus anionic polymermolar ratios of 5.5:1 and 10:1 (as shown in the bars labeledn/(p+2c)=5.5 or 10), with or without addition of HTP. Addition of PDGAdid not impair complexation kinetics at any of the molar ratios tested.

Effects of including a cationic polymer and cationic polypeptide onpolyplex destabilization were also determined, as shown in FIG. 5.Polyplex nanoparticles of DNA and cationic polypeptides (PLR with orwithout HTP, or PEI with HTP) with [(PDGA) carboxylate(c):(DNA)phosphate(p)] molar ratios varying from 0 to 100 were complexed asdescribed, compared to DNA or EtBr alone as controls, and the effects ofdestabilization (as indicated by increased fluorescence) was determined.In the absence of HTP, addition of PDGA did not lead to polyplexdestabilization. However, in the presence of HTP, adding molar ratios ofPDGA to DNA of 20 and above led to polyplex destabilization. Theseresults indicate a surprising synergistic effect of cationic polypeptideand anionic polymer on complex destabilization. Cationic polypeptideincorporation, and/or inclusion of cationic constituents of disparatemolecular weights or sizes, into a nanoparticle polyplex core maybeneficially enhance the ability of a cationic polymer to promotedissociation and release of the polynucleotide payload from the polyplexand its other constituents.

Dynamic light scattering (BRAND) was used to determine the hydrodynamicradii of nanoparticles at various stages of formation. Nanoparticlescontaining core polyplexes with plasmid DNA, PLR, PDGA, and HTP, at amolar ratio of [amide]:[(phosphate)] of 5.5:1 were complexed asdescribed. Some polyplex cores were further coated with silica asdescribed. And some silica-coated polyplexes were further layered withcationic polymer (SEQ ID NO: 4) as described. 30-60 minutes ofmeasurements were obtained following initial core formation of ternarycomplexes, silica coating of cores, and cationic polymer-coating ofsilica-coated cores. FIG. 6 is a graph showing diameters ofnanoparticles. Uncoated polyplex cores and polyplex cores coated withsilica were approximately 70-150 nm in diameter on average. In otherembodiments, polyplex cores and silica-coated polyplex cores may bewithin a range of 100-170 nm in average diameter. Adding a cationicpolymer coating to the silica coating yielded a nanoparticle with anaverage diameter of approximately 170 nm. In other embodiments,silica-coated polyplex cores with an additional layer of cationicpolymer attached to the outer layer of silica may be within a range ofapproximately 80-200 nm in average diameter.

Cellular uptake of nanoparticles was also determined. Fluoresceinisothiocyanate (FITC) was covalently conjugated to amines of PEI (25 kDalinear) and PLR (29 kDa) such that the molar ratio of amines to FITC was100:1. The reaction was performed in darkness at room temperature forfour hours in equal volumes of water and DMSO. In order to establishconjugation, a 0.05% w/v 500 uL solution of each fluorescently modifiedpolymer was centrifuged in a 10 kDa Nanosep® filter and the eluate'sfluorescence intensity (485 ex./520 em.) was compared to the unfilteredpolymer solution as well as water. mCherry plasmid (Addgene) wasincluded in nanoparticles to permit fluorescent detection ofplasmid-driven expression.

MC3T3 murine osteoblasts were cultured on polystyrene T-75 tissueculture plastic flasks (Corning, Calif., USA). Dulbecco's modified eaglemedium supplemented with 10% Fetal Bovine Serum (Thermo FisherScientific, VA, USA) was used for osteoblasts along with 1%penicillin/streptomycin (Invitrogen, NY, USA). Xylenol orange was addedto the cell culture media from day 15 to day 25 after initiation of cellculture. At day 25 cells were fixed and assayed for mineralization. FormCherry plasmid delivery using FITC-modified nanoparticles, osteoblastswere plated at 1000 cells/well in 96-well plates and allowed to adherefor 12-16 hours in antibiotic-free DMEM containing 10% FBS. Immediatelybefore transfection, medium was replaced with equal volumes ofOptiMEM-suspended nanoparticles and DMEM containing 10% FBS.

All complexes were FITC-labeled and subjected to qualitative observationof fluorescence intensity (488/520 ex./em.) before transfection.96-well-plated osteoblasts (1000 cells/well) were transfected with 200ng of plasmids in triplicates for each binary (plasmid and cationicpolymer), ternary (plasmid and cationic polymer, plus anionic polymer orcationic polypeptide), and quaternary (plasmid, cationic polymer,anionic polymer, and cationic polypeptide) complex as well as itssilica-coated counterpart, with 1 control and 8 experimental sets (n=3)in total. 5% serum was used in order to study effects of serum onextracellular properties of aggregation.

At 30-hours post-transfection, bimodal fluorescent imaging allowed forsimultaneous observation of FITC-labeled nanoparticles (488 ex./520 em.)and the mCherry gene expression that they were responsible for (633ex./680 em.). A minimum of 20 cells were observed at different locationsin each well and representative images were obtained. ImageJ was used toprocess the overlaid images and combine phase-contrast, 488/520 and633/680 channels.

Photomicrographs demonstrating cellular uptake are shown in FIG. 7.Circles in FIG. 7 indicate where high levels of nuclear localization isapparent. Silica-coated binary nanoparticles show burst releaseproperties (i.e., nuclear localization is not apparent in theDNA-PLR+silica samples). Inclusion of PDGA in polyplex cores causesprolonged release of plasmid within cell nuclei. This effect of PDGA tocause prolonged release was surprising in light of literature suggestingthe opposite: that including cationic polymers in nanoparticlepolyplexes would hasten, and shorten the duration of, dissociation ofpolynucleotide payload from other polyplex constituents. Addition of HTPalso causes extensive nuclear localization.

Further coating of silica-coated nanoparticles (DNA-HTP-PDGA-PLR+Si)with poly(arginine) (SEQ ID NO: 4) causes nanoparticles to be stable inserum and causes extended residence of nanoparticle payload withincells. FIG. 8. is photomicrographs showing cellular uptake and retentionof silica-coated FITC-conjugated polyplex cores, to which an additionallayer of poly(L-arginine) (SEQ ID NO: 4) has been added, by MC3T3 murineosteoblasts, in accordance with the present invention. Unlike forsilica-coated nanoparticles shown in FIG. 7, no aggregation ofnanoparticles containing an additional layer of cationic polymers on theoutside of the silica coating is observable in FIG. 8, indicating thatsuch nanoparticles remain stable in serum. Furthermore, thesenanoparticles are observed to display extended residence within the cellnucleus such that fluorescence qualitatively peaks within approximately1.5 days and detectable fluorescence was sustained through 14 days.

Layering silica-coated polyplex cores with polymers specificallydirected to bind to particular cell types can further enhance uptake.Associating ligands for cellular receptors with the surface of ananoparticle can enhance affinity of the nanoparticle for cells thatexpress such receptors and increase transfection of such cells. As oneexample in accordance with the present invention, silica-coatedpolyplexes were coated with VEGF (SEQ ID NO: 5), a high-affinity ligandfor VEGF receptors, which are expressed at high levels by humanumbilical vein endothelial cells (HUVECs). HUVECs were incubated withsilica-coated FITC-conjugated polyplexes with poly(L-arginine) (SEQ IDNO: 4) or human VEGF (SEQ ID NO: 5) attached to the outer surface of thesilica coating for 40 min before being washed twice with PBS thenresuspended in DMEM (10% FBS). Cells were imaged 4 hrs later. After thisshort incubation period, only low levels of transfection withnanoparticles containing a poly(L-arginine) layer attached to theexternal silica surface (FIG. 9A) was observed, whereas coating withVEGF instead of poly(L-arginine) resulted in significantly greatercellular internalization at this four-hour time point. A skilled artisanwould recognize that virtually any other cell type may also betransfected by nanoparticles in accordance with the present invention,and that a layer of polymers may be attached to the outer layer ofsilica-coated polyplex cores to promote or otherwise influence thiseffect. Such a person would also comprehend that other means ofcontacting cells with nanoparticles to effect such outcomes, such asi.p., i.v., i.m. or s.c. or other injection or transdermaladministration or via suppository to, or ingestion or oral or nasalinhalation by, a human or animal, or contact with explanted tissue orcells or stem cells, would also be included within the presentinvention.

In another aspect of the invention, a polynucleotide encoding a nucleasemay be incorporated into the nanoparticle polyplex core. As onenonlimiting example, a polynucleotide that encodes and drives expressionof a TALEN (Transcription Factor-Like Effector Nucleases) may beincluded in the nanoparticle. Like Zinc Finger Nucleases, TALENs utilizea modular DNA binding motif (TALE) that can be modified to introduce newDNA binding specificities and even nucleases (TALEN). TALEs consist ofmultiple repeat variable diresidues (RVDs) which each specify binding toa single nucleotide. TALE arrays are made by stringing together RVDs ina specific order to provide specificity and binding affinity to desiredDNA sequences. Commonly, these genome-splicing tools are engineered byfusing non-specific cleavage domains, such as FokI nucleases, to TALEs.TALEN assembly protocols are available that allow assembly of theserepetitive sequences, including an open source assembly method known asGolden Gate.

In another aspect of the present invention, nanoparticles may bedesigned and used in a manner to regulate expression of signalingmolecules to alter cellular function. For example, sequences ofchromosomal DNA may be deleted or altered to generate cellular or animalmodels of disease states or treatments therefor, or to treat diseasestates or otherwise enhance human health. One nonlimiting example of aprotein whose expression may be modified in accordance with the presentinvention is sclerostin (SOST). SOST binding to the LRPS/6 receptorinhibits Wnt signaling, perhaps via feedback systems between Wnt3A,Wnt7B, Wnt10A, sclerostin, β-catenin, LEF1, and TCF1. Desuppressingthese cascades via removal of sclerostin may result in significantlyincreased mineralization activity.

Osteoprogenitor (OPG) and RANKL are also expected to play a responsiverole to SOST deletion, where RANKL expresses itself as a receptor forpromoting osteoclastogenesis via osteoclast-linked RANK or ODF(osteoclast differentiation factor) binding, and OPG bindsantagonistically to RANKL. Thus, the ratio between OPG and RANKL is adeterminant of the relationship between bone formation and resorption.However, single cultures of osteoblasts will communicate through otherforms of paracrine signaling and this ratio should be more reflective ofbehavior of altered cells in co-culture with osteoclasts or in vivo.

In another aspect of the present invention, a nanoparticle may bedesigned so as to allow transfection with a TALEN that may disruptexpression of SOST and consequently generate a high bone-mass phenotype.As one example, TALENS may be engineered to specifically bind to loci inthe SOST gene and create double-stranded breaks in the genome to disrupttranscription or translation and reduce SOST expression. As a furtherexample, a nanoparticle may contain plasmids that encode two TALENs thatcreate double-stranded breaks on either side of the chromosomal locus ofthe start codon for SOST. Repair of endogenous genomic DNA followingexcision of the sequence encoding the start codon may result intranscription of sclerostin mRNA lacking the start codon that cannot beproperly translated into SOST protein, thereby driving down SOSTexpression and activity. A diagrammatic representation of this model isshown in FIG. 10, where a “left” TALEN and “right” TALEN bind to andcleave sites on opposite sides of the SOST start codon locus. As oneexample, a left TALEN may have the sequence represented by SEQ ID NO: 6,and a right TALEN may have the sequence represented by SEQ ID NO: 7. Ananoparticle may comprise an expression plasmid, such as pUC19 (GenbankAccession Number L09137 X02514), into which a nucleotide sequence thatencodes a right or left TALEN, such as those represented by SEQ ID NO: 6and SEQ ID NO: 7, has been subcloned so as to drive cellular expressionof the encoded TALEN. A nanoparticle may also include combinations ofexpression plasmids that comprise sequences that encode left and rightTALENs.

A nanoparticle may also comprise other TALEN sequences, targeting SOSTor any other gene of interest, and also may comprise other expressionvectors, in accordance with the present invention. A nanoparticle maycomprise other types of polynucleotides or analogs thereof, such asspecies of RNA or DNA including mRNA, siRNA, miRNA, aptamers, shRNA,AAV-derived nucleic acids, morpholeno RNA, peptoid and peptide nucleicacids, cDNA, DNA origami, DNA and RNA with synthetic nucleotides, DNAand RNA with predefined secondary structures, CRISPR sequences, andmultimers and oligomers, and any combination of the foregoing, inaccordance with the present invention. In another example, ananoparticle may comprise polynucleotides whose sequence may encodeother products such as any protein or polypeptide whose expression isdesired. A skilled artisan would recognize that the foregoing examplesare in accordance with the present invention and may be encompassed byclaims thereto.

Following transfection of MC3T3 murine osteoblasts with nanoparticlesdesigned to knock down SOST expression in accordance with the presentinvention, ELISA and quantitative real-time PCR (qPCR) assays wereperformed on cell lysate and supernatant fractions. FIGS. 11A-11C aregraphs demonstrating the effectiveness of different amounts (800 ng,1600 ng, or 2500 ng) of nanoparticles (NP) containing expressionplasmids comprising nucleotide sequences that encode left (SEQ ID NO: 6)and right (SEQ ID NO: 7) SOST TALENs, in accordance with the presentinvention, in modulating SOST expression and β-catenin expression over aperiod of up to over 20 days following transfection. For comparison,other cells were transfected with mRNA encoding the same TALENS usingLipofectamine, a known agent for cellular transfection. As shown inFIGS. 11A-11C, intracellular and extracellular SOST levels weresuppressed for at least several weeks following transfection withnanoparticles in accordance with the present invention, whereasβ-catenin expression was concomitantly up-regulated, signifyingeffectiveness of the nanoparticles in downregulating SOST expression andactivity.

qPCR was also performed to determine whether down-regulation of SOSTexpression with nanoparticles in accordance with the present inventionmay have downstream effects on other components of the relevantsignaling cascade. Cells were transfected as described above. Results onexpression of numerous components of the signaling pathway (SOST,β-catenin, TCF1, LEF1, Wnt3A, Wnt7B, Wnt10b, OPG, and RANKL), at 5, 14,and 21 days after transfection with different amounts of nanoparticlesas indicated, are shown in FIGS. 12A-12F. For comparison, other cellswere transfected with mRNA encoding the same TALENS using Lipofectamine.The real time PCR results showed a greater up regulation of Wntresponsive genes in the cell lines transfected with nanoparticlesdelivering SOST TALENS as compared to the SOST TALENS delivered byLipofectamine by up to 2 to 6 times as a response to knockdown of theWnt signaling inhibitor sclerostin.

TCF/LEF-1-mediated transcription may also be upregulated followingknockdown of SOST expression in accordance with the present invention.MC3T3-E1 cells were transfected with TOPflash and control FOPflashluciferase reporter plasmid constructs (Addgene#12456 and 12457) thatcontain TCF/LEF-1 binding sites. The cells were plated at the density of5000 cells/well of the 8-well labtek chamber slides and transfected with1 ug of TOPflash and FOPflash plasmid separately. To control for theefficiency of transfection a control plasmid Renilla (Promega) was used.FIG. 13 is photomicrographs showing upregulation of TCF/LEF-1-mediatedtranscription for 21 days following tranfection with nanoparticlescontaining plasmids encoding SOST-directed TALENS, in accordance withthe present invention, consistent with an upregulation of TCF/LEF-1expression and activity following transfection with the inventednanoparticles.

Knockdown of SOST expression in accordance with the present inventionmay also increase mineralization in stromal bone marrow cells andosteoblasts. Mineralization was quantified by two separate methods,first based on image thresholding of xylenol-orange-labeled vitalcultures using MATLAB (Mathworks, Natick, Mass.), and second by atomicabsorption spectroscopy (AAS). For the xylenol orange threshold, imagesof both phase and fluorescence (with Texas Red Filter Set) were taken infive adjacent regions of wells, and then stitched into a larger 8-bitimage (4×, Nikon Ti-100). The phase channel was subtracted from thefluorescence, and a threshold was set to half the level between thebackground and signal (−6 dB). The number of pixels above the thresholdwere counted and used to express the percentage of mineralized area ineach well. The combination of phase and fluorescence allowed for unboundxylenol orange to be distinguished, whereas the use of decibel levelsallowed for correction of the varied background levels in each image.

Mineralization was also quantified by atomic absorption with an atomicabsorption spectrometer (AA-Perkin Elmer, Mass.). Each well was preparedby adding 0.5 mL of 10% nitric acid, and the resultant calcium contentwas measured relative to a standard curve and compared between groups.Care was taken to minimize interference due to ionized calciumprecipitating with phosphate phases, so a large excess of potassium andlanthanum ions was added to each well.

FIGS. 14A-14C show the effects of transfection with nanoparticles inaccordance with the present invention on mineralization following SOSTknockdown. FIG. 14A is photomicrographs of staining of the mineralizedmatrix formed 25 days after SOST knockdown. Stromal cells are shown inpanels A-C, wherein panel A show control cells, panel B shows cellstransfected via Lipofectamine, and panel C shows cells transfected withnanoparticles containing plasmids encoding SOST-directed TALENs asdescribed and in accordance with the present invention. MC3T3-E1osteoblast cells are shown in panels D-G, wherein panel D show controlcells, and panels E-G show cells transfected with nanoparticlescontaining plasmids encoding SOST-directed TALENs as described at dosesof 800 ng, 1600 ng, and 2500 ng, respectively, in accordance with thepresent invention. FIGS. 14B and 14C are graphs showing quantificationof mineralization. FIGS. 14A-C demonstrate increased calciumconcentration in stromal bone marrow cells and osteoblasts followingtransfection with SOST-targetting TALENS via nanoparticles in accordancewith the present invention, further confirming the effectiveness of thistechnique of modifying the cellular expression and activity of genes anddownstream signaling pathways.

While several aspects of the present invention have been described anddepicted herein, alternative aspects may be effected by those skilled inthe art to accomplish the same objectives. Accordingly, it is intendedby the appended claims to cover all such alternative aspects as fallwithin the true spirit and scope of the invention.

1. A nanoparticle comprising: a core polyplex and a silica coatingthereon; wherein said core polyplex comprises an anionic polymer, acationic polymer, a cationic polypeptide, and a polynucleotide.
 2. Thenanoparticle of claim 1 wherein the anionic polymer is poly(D-glutamicacid).
 3. The nanoparticle of claim 1 wherein the cationic polymer isselected from the group consisting of poly(ethylenimine) andpoly(L-arginine).
 4. The nanoparticle of claim 1 wherein the cationicpolypeptide is a histone tail peptide.
 5. The nanoparticle of claim 4wherein the histone tail peptide is human H3 histone tail peptide. 6.The nanoparticle of claim 1 wherein the anionic polymer ispoly(D-glutamic acid), the cationic polymer is selected from the groupconsisting of poly(ethylenimine) and poly(L-arginine), and the cationicpolypeptide is a histone tail peptide.
 7. The nanoparticle of claim 6wherein the polynucleotide comprises a nucleotide sequence that encodesa nuclease.
 8. The nanoparticle of claim 7 wherein the nuclease is aTALEN.
 9. The nanoparticle of claim 8 wherein the TALEN is capable ofinducing a break at a site-specific locus of DNA, wherein the breakresults in a change of expression of a protein encoded by a gene. 10.The nanoparticle of claim 9 wherein the change is a decrease and thegene encodes a sclerostin protein.
 11. A nanoparticle of claim 6,further comprising a polymer attached to an outer surface of said silicacoating.
 12. A nanoparticle of claim 11, wherein said polymer attachedto an outer surface of said silica coating comprises poly(L-arginine) ora vasoactive endothelial growth factor peptide.
 13. A method ofmodifying intracellular polynucleotides comprising; contacting a cellwith a nanoparticle, wherein said nanoparticle comprises a core polyplexand a silica coating thereon; wherein said core polyplex comprises ananionic polymer, a cationic polymer, a cationic polypeptide, and apolynucleotide.
 14. The method of claim 13 wherein the anionic polymeris poly(D-glutamic acid).
 15. The method of claim 13 wherein thecationic polymer is selected from the group consisting ofpoly(ethylenimine) and poly(L-arginine).
 16. The method of claim 13wherein the cationic polypeptide is a histone tail peptide.
 17. Themethod of claim 16 wherein the histone tail peptide is human H3 histonetail peptide.
 18. The method of claim 13 wherein the anionic polymer ispoly(D-glutamic acid), the cationic polymer is selected from the groupconsisting of poly(ethylenimine) and poly(L-arginine), and the cationicpolypeptide is a histone tail peptide.
 19. The method of claim 18wherein the polynucleotide comprises a nucleotide sequence that encodesa nuclease.
 20. The method of claim 19 wherein the nuclease is a TALEN.21. The method of claim 20 wherein the TALEN is capable of inducing abreak at a site-specific locus of DNA, wherein the break results in achange of expression of a protein encoded by a gene.
 22. The method ofclaim 21 wherein the change is a decrease and the gene encodes asclerostin protein.
 23. The method of claim 18, further comprising apolymer attached to an outer surface of said silica coating.
 24. Themethod of claim 23, wherein said polymer attached to an outer surface ofsaid silica coating comprises poly(L-arginine) or a vasoactiveendothelial growth factor peptide.